Methods for screening for histone deacetylase activity and for identifying histone deacetylase inhibitors

ABSTRACT

The present invention relates to novel methods for screening for histone deacetylase enzyme activity in a test sample. The present invention further relates to novel methods for screening potential inhibitors of histone deacetylase enzymes.

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/383,308, filed May 23, 2002, the contents of which ishereby incorporated by reference in its entirety.

[0002] This work was supported in part by grant number GM65539 from theNational Institutes of Health. The United States government may haverights in this invention by virtue of this support.

BACKGROUND OF THE INVENTION

[0003] 1. Field of Invention

[0004] The present invention relates to novel methods for screening forhistone deacetylase enzyme activity in a test sample. The presentinvention further relates to novel methods for screening potentialinhibitors of histone deacetylase enzymes.

[0005] 2. Background

[0006] Histones and Gene Expression. In eukaryotic cells, nuclear DNAassociates with histones to form a compact complex called chromatin. Thehistones constitute a family of basic proteins which are generallyhighly conserved across eukaryotic species. The core histones, termedH2A, H2B, H3, and H4, associate to form a protein core. Histones are theprotein portion of a protein-DNA complex termed the nucleosome. DNAwinds around this protein core, with the basic amino acids of thehistones interacting with the negatively charged phosphate groups of theDNA. Nucleosomes structurally organize chromosomal DNA to formchromatin. The repeating structural motif of chromatin is the nucleosomeparticle, which is comprised of approximately 146 base pairs of DNAwrapped around a histone core.

[0007] Histones are subject to post-translational acetylation of theε-amino groups of N-terminal lysine residues, a reaction that iscatalyzed by histone acetyl transferase. The degree of interactionbetween histones and DNA varies between regions undergoing transcriptionand regions not being transcribed in normal cells. The histones inchromatin regions under active transcription are oftenpost-translationally modified with acetyl groups covalently attached tospecific lysine residues, which has been both positively and negativelycorrelated with gene activity. Discovered nearly 40 years ago, it hasonly recently become clear that acetylation of the ε-amino group ofspecific lysine residues within the positively charged N-terminal tailof core histones H2A, H2B, H3, and H4 results in localized chromatinrelaxation and a change in both histone-DNA and histone-nonhistoneprotein interaction.

[0008] Histone deacetylase (hereinafter “HDAC”) and histoneacetyltransferase together control the net level of acetylation ofhistones and maintain the delicate dynamic equilibrium in theacetylation level of nucleosomal histones. In general, acetylationactivity is correlated with transcriptional activation, whereasdeacetylation activity is accompanied by transcriptional repression.Acetylation neutralizes the positive charge of the lysine side chain,and is thought to impact chromatin structure. Access of transcriptionfactors to chromatin templates is enhanced by histone hyperacetylation.An enrichment in underacetylated histone H4 has been found intranscriptionally silent regions of the genome.

[0009] Histone acetylation is a reversible modification, withdeacetylation being catalyzed by members of the histone deacetylasefamily of enzymes. Histone deacetylases are responsible forde-acetylation and may be localized to DNA targeted for repression byother proteins that associate with HDAC and specifically bind regulatoryelements in genes. HDACs are divided into two classes, Class Irepresented by yeast Rpd3-like proteins, and Class II represented byyeast Hda1-like proteins. Human HDAC1, HDAC2, HDAC3, and HDAC8 proteinsare members of the Class I group of HDACs. Human HDAC4, HDAC5, HDAC6,and HDAC7 are members of the Class II group of HDACs.

[0010] Inhibition of the action of histone deacetylase results in theaccumulation of hyperacetylated histones, which in turn is implicated ina variety of cellular responses, including altered gene expression, celldifferentiation, and cell-cycle arrest. Hyperacetylated histones arethought to adopt a chromatin structure that allows other proteins toactivate DNA transcription. Inactive genes are associated withhypoacetylated histones, and removal of the acetyl groups from histonesin normally active chromatin will repress transcription in that region.The reversible acetylation of histones is crucial for thetranscriptional regulation of gene expression in eukaryotic cells.

[0011] HDAC activity is inhibited by trichostatin A (TSA), a naturalproduct isolated from Streptomyces hygroscopicus, and by a syntheticcompound, suberoylanilide hydroxamic acid (SAHA). TSA arrestsdevelopment of rat fibroblasts at the G₁ and G₂ phases of the cellcycle, implicating HDAC in cell cycle regulation. TSA and SAHA inhibitcell growth, induce terminal differentiation, and prevent the formationof tumors in mice. These findings suggest that inhibition of HDACactivity represents a novel approach for intervening in cell cycleregulation and that HDAC inhibitors have great therapeutic potential inthe treatment of cell proliferative diseases or conditions. To date,only a few inhibitors of histone deacetylase are known in the art. Thereis thus a great need to identify additional HDAC inhibitors and toidentify the structural features required for potent HDAC inhibitoryactivity.

[0012] Coumarin Derivatives. 2-H-1-benzopyran-2-one, commonly known asCoumarin, is the parent organic compound of a class of naturallyoccurring phytochemicals found in many plant species. An oxygenheterocycle, it is best known for its fragrance, described as avanilla-like odor or the aroma of freshly mowed hay. Identified in the1820s, coumarin has been synthesized in the laboratory since 1868 andused to make perfumes and flavorings. Chemically, coumarin can occureither free or combined with glucose to produce a coumarin glycosidederivative. Medically, other coumarin glycoside derivatives have beenshown to have blood-thinning, anti-fungicidal, and anti-tumoractivities. Dicumarol, a coumarin glycoside better known as warfarin, isa commonly used oral anticoagulant. It undergoes very extensivemetabolism along two major pathways, 7-hydroxylation and ring-opening toortho-hydroxyphenylacetaldehyde. The relative extent of these two majorpathways is highly variable between species. Ring-opening predominatesin rodents, while 7-hydroxylation is particularly evident in humans.

[0013] Coumarin is the basic structure of numerous naturally occurringcompounds with important and diverse physiological activities. More thana thousand coumarin derivatives have been described, varying from simplecoumarins containing alkyl and hydroxyl side chains to complex coumarinswith benzoyl, furanoyl, pyranoyl, or alkylphosphorothionyl substituents.The structures of coumarin and some of its most common pharmaceuticalderivatives are shown below.

[0014] Additionally, a number of coumarin derivatives have fluorescenceproperties and are used as dye molecules; an example are the7-aminocoumarins, which exhibit characteristic, molecule-specific peakabsorption and fluorescence emission spectra. For example, a commonlaser dye, 7-diethylamino-4-methylcoumarin or Coumarin 1, has a peakabsorption wavelength of about 373 nm and a peak emission wavelength ofabout 460 nm. Other common exemplary coumarin derivatives with knownfluorescence properties include Coumarin 6, 30, 35, 102, 120, 138, 151,152, and 153. The fluorescence properties of a large number of coumarinderivatives, having fluorescence emission spectra over a wide range, areknown of to those of ordinary skill in the art.

[0015] Prior Art HDAC Assays. The characterization of HDAC activity hasbeen problematic for many reasons. First, it is difficult to obtainsufficient quantities of pure enzyme that retain catalytic activity.Histone deacetylases are zinc-dependent enzymes, and many of the methodsestablished for purifying recombinant proteins with an engineeredaffinity tag require the use of chelators that may strip the enzyme ofits metal. Second, all previously established in vitro activity assaysare discontinuous or require the use of radioactivity. The acetateextraction method relies on the use of a [3H]-acetylated histone peptidesubstrate corresponding to the sequence of a histone tail. The HDACenzyme is incubated with the substrate, and the reaction quenched withhydrochloric and acetic acid. Next the released [3H] acetic acid isextracted with ethyl acetate and quantified by scintillation counting.It has been shown that the solubility of acetic acid in ethyl acetate islimiting, causing an underestimate of 3H acetate removed.

[0016] The discovery of novel HDAC inhibitors as new drugs fortranscription therapy and cancer chemoprevention is currently obstructedby the lack of suitable assay systems. One widely distributed assay ofHDAC activity depends on the incubation of the enzyme withacetate-radiolabeled histones or peptide substrates, followed byextraction with organic solvents such as ethyl acetate and then thequantification of the released radiolabeled acetic acid by liquidscintillation count. 3H-histones are obtained by a laborious procedurethat relies on the sacrifice of animals. The degree of acetylation ofprelabeled core histones changes within different preparations and it istherefore difficult to standardize the substrate properties.

[0017] Similarly, labeled oligopeptides are synthesized by solid-phasetechnology, and postlabeling HPLC purification is required. Althoughclassical radioactive assays have been successfully used to measure HDACactivities from various sources, the need to separate product fromsubstrate limits assay throughput. In addition, the use of scintillationcocktails makes these assays costly in terms of time, labor, andradioactive waste. Hence, assays of this type are not readily amenableto automation and high-throughput screening.

[0018] A principally nonisotopic method for the determination of HDACactivity relied on immunoblotting of hyperacetylated histones. However,this approach has the drawback that rather than measuring enzymeactivity in the presence or absence of inhibitor, these immunoblottingprocedures more resembled functional tools that are not suited for assaythroughput. Another nonisotopic assay for HDAC activity used(N-(4-methyl-7-coumarinyl)-(tert-butyloxy-carbonyl)-acetyllysinamide) asa substrate. Unfortunately, formation of the deacetylated product ismonitored by HPLC and fluorescence detection after extraction with ethylacetate. As a result, these prior art assays are not well suited forhigh-throughput screening.

[0019] In addition, the substrates used in the prior art do not wellresemble acetylated lysine residues in the original context of histones.Recent improvements of this assay include: (1) fluorescence-labeledoctapeptide substrates that bear some closer resemblance to the nativesubstrate; and the introduction of an internal standard for thequantification of fluorescence substrate by HPLC. Although the prior artassays have been used with limited success, including for the study oftime- and site-dependent deacetylation, they still remain not readilyamenable to automation and high-throughput screening due to the requiredseparation steps.

[0020] Other assays for histone deacetylase activity can be used as apreliminary screen to select candidates for other differentiationagents. For example, U.S. Pat. No. 5,922,837 discloses an assay usingtritiated desmethoxyapicidin and a parasite or chick liver S100 solutionas a source of deacetylase activity. The candidate compound is added tothe reaction mixture, and tritium release is measured using a filtermethod.

[0021] Nare et al., Anal. Biochem. 267:390 (1999) have developed ascintillation proximity assay using a peptide from histone H4, withlysine ε-amino groups acetylated with tritium, and bound to an SPA beadthat scintillates proportionately to the amount of proximal tritium.Histone deacetylase activity, obtained from extracts of HeLa cellnuclei, releases the labeled acetyl groups and decreases scintillation,and the presence of a deacetylase inhibitor maintains scintillation.

[0022] Hoffman et al., Nucl. Acids Res. 27:2057 (1999) describes anon-isotopic assay for histone deacetylase activity. A fluorescentsubstrate has been developed that is an aminocoumarine derivative ofΩ-acetylated lysine. This permits quantitation of substrate in thenanomolar concentration range, which allows for high throughputscreening of histone deacetylase inhibitors.

[0023] Manfred Jung and colleagues have developed a number of HDACassays that do not rely on radioactivity. The first non-isotopic assayutilizes a fluorescent derivative of ε-acetyl lysine,(N-(4-methyl-7-coumarinyl)-α-(tert-butyloxy-carbonyl)-W-acetyllysinamide),which can be quantified using a reverse-phase HPLC system with afluorescence detector. An internal standard was later employed toimprove the accuracy of the assay. Fluorescein-labeled octapeptides weredesigned to more closely resemble the native HDAC substrate andconstructed for use in another HPLC and fluorescence detection basedassay. Their studies revealed a preference for the C-terminalacetyl-lysine position, but this finding may be due to steric hindranceassociated with the N-terminal fluorescein group.

[0024] Recently, a commercially developed kit, which comprises aproprietary acetylated lysine side chain substrate, was introduced bythe Plymouth Meeting, Pennsylvania company BIOMOL Research Laboratories,Inc., for high-throughput screening of HDAC activity in a 96-well plateformat. In this assay, HDAC enzyme is incubated with the substrate, thereaction is quenched with a proprietary developer solution, and theresulting fluorescent signal is quantified using a fluorescencemicrotiter plate reader.

[0025] U.S. Pat. No. 6,428,983 issued Aug. 6, 2002 to Dulski, et al.discloses a method for identifying compounds that are antiprotozoalagents by determining whether a test compound or natural product extractinhibits the action of protozoal histone deacetylase, said methodcomprising: (a) contacting a protozoal histone deacetylase, or anextract containing protozoal histone deacetylase with a known amount ofa labeled compound that interacts with a protozoal histone deacetylasein the presence or absence of a known dilution of a test compound or anatural product extract; and (b) quantitating the percent inhibition ofinteraction of said labeled compound by the difference in the enzymeactivities in the presence and absence of the inhibitor test compound ornatural product extract. Dulski, et al. disclose that substrates forhistone deacetylase may be acetylated histones, or a labeled acetylatedpeptide fragment derived therefrom such asAcGly-Ala-Lys(ε-Ac)-Arg-His-Arg-Lys(ε-Ac)-ValNH₂, or other synthetic ornaturally occurring substrates. Examples of known compounds that bind tohistone deacetylase are known inhibitors such as α-butyrate,trichostatin, trapoxin A, and other inhibitors.

[0026] Wegener D, Wirsching F, Riester D, Schwienhorst A, A fluorogenichistone deacetylase assay well suited for high-throughput activityscreening, Chem. Biol. Jan;10(1):61-8 (2003), which was published afterthe priority date of this application, discloses a fluorogenic assay forHDAC activity for expediting studies of HDAC in transcriptionalregulation and in vitro screening for drug discovery. In that work,fluorogenic substrates of HDACs were synthesized with anepsilon-acetylated lysyl moiety and an adjacent MCA moiety at the Cterminus of the peptide chain. Upon deacetylation of the acetylatedlysyl moiety, molecules became substrates for trypsin, which releasedhighly fluorescent AMC molecules in a subsequent step of the assay. Thefluorescence increased in direct proportion to the amount ofdeacetylated substrate molecules, i.e., HDAC activity. The primarydrawback to fluorescent assays is that, although they are suitable forhigh-throughput screening and they alleviate the need for radioactiveisotopes, they are all still discontinuous.

[0027] Thus, histone deacetylases are important enzymes for thetranscriptional regulation of gene expression in eukaryotic cells.Recent findings suggest that HDACs could be key targets forchemotherapeutic intervention in malignant diseases. Compounds andcompositions identified by the inventive methods are expected to beinhibitors of histone deacetylase, and are expected to inhibit cellproliferation in a contacted cell by growth retardation, growth arrest,programmed cell death, or necrotic cell death.

[0028] A convenient, sensitive, high-throughput assay for HDAC activitywould therefore expedite studies of HDAC in transcriptional regulationand greatly improve in vitro screening for drug discovery. In responseto this need, the inventive subject matter provides novel continuousassays for HDAC activity and provides for improved methods for selectionof HDAC inhibitors. In particular, the inventive subject matter providescontinuous, non-isotopic, spectrophotomeric—both fluorescent andchromogenic—assays for HDAC activity, and will greatly streamline theselection process for HDAC inhibitors, both generally and targeted tospecific histone deacetylase enzymes.

SUMMARY OF THE INVENTION

[0029] The present invention relates to a method for screening forhistone deacetylase enzyme activity in a test sample, comprising thesteps of:

[0030] i) contacting said test sample with a liquid screening solutioncomprising a compound of Formula I:

[0031]  wherein:

[0032] R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of:

[0033] hydrogen,

[0034] hydroxyl,

[0035] carbonyl,

[0036] acetyl,

[0037] Ar,

[0038] straight or branched chain C₁-C₉ alkyl,

[0039] straight or branched chain C₁-C₉ alkyl substituted with one ormore halo, trifluoromethyl, nitro, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy,C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0040] straight or branched chain C₂-C₉ alkenyl or alkynyl, and

[0041] straight or branched chain C₂-C₉ alkenyl or alkynyl substitutedwith one or more halo, trifluoromethyl, nitro, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy,C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0042] provided that at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈must be —O—C(O)—R₉, wherein R₉ is straight or branched chain C₁-C₃alkyl; and

[0043] Ar is a mono-, bi- or tricyclic, carbo- or heterocyclic ring,wherein the ring is either unsubstituted or substituted in one or moreposition(s) with halo, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straightor branched chain alkyl or alkenyl, C₁-C₄ alkoxy, C₁-C₄ alkenyloxy,phenoxy, benzyloxy, or amino; wherein the individual ring sizes are 5-6members; and wherein the heterocyclic ring contains 1-6 heteroatom(s)selected from the group consisting of O, N, and S; and

[0044] ii) assaying for fluorescence produced by a product of histonedeacetylase enzyme activity in said liquid screening solution.

[0045] The present invention further relates to a method for screeningfor inhibitors of histone deacetylase enzyme activity, comprising thesteps of:

[0046] i) contacting an inhibitor candidate compound or composition witha liquid screening solution comprising a histone deacetylase enzyme anda compound of Formula I:

[0047]  wherein:

[0048] R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of:

[0049] hydrogen,

[0050] hydroxyl,

[0051] carbonyl,

[0052] acetyl,

[0053] Ar,

[0054] straight or branched chain C₁-C₉ alkyl,

[0055] straight or branched chain C₁-C₉ alkyl substituted with one ormore halo, trifluoromethyl, nitro, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy,C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0056] straight or branched chain C₂-C₉ alkenyl or alkynyl, and

[0057] straight or branched chain C₂-C₉ alkenyl or alkynyl substitutedwith one or more halo, trifluoromethyl, nitro, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy,C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0058] provided that at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈must be —O—C(O)—R₉, wherein R₉ is straight or branched chain C₁-C₃alkyl; and

[0059] Ar is a mono-, bi- or tricyclic, carbo- or heterocyclic ring,wherein the ring is either unsubstituted or substituted in one or moreposition(s) with halo, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straightor branched chain alkyl or alkenyl, C₁-C₄ alkoxy, C₁-C₄ alkenyloxy,phenoxy, benzyloxy, or amino; wherein the individual ring sizes are 5-6members; and wherein the heterocyclic ring contains 1-6 heteroatom(s)selected from the group consisting of O, N, and S; and

[0060] ii) assaying for fluorescence produced by a product of histonedeacetylase enzyme activity in said reaction solution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061]FIG. 1 is a drawing which depicts the inventive process ofmonitoring the hydrolysis of 7AC to 7-hydroxycoumarin.

[0062]FIG. 2A is a drawing which depicts a MBP-Hos3 plasmid map.

[0063]FIG. 2B is a drawing which depicts the junction between Hos3 andMBP showing the TEV protease cleavage site in a MBP-Hos3 plasmid map.

[0064]FIG. 2C is a photograph which depicts an affinity purification gelof MBP-Hos3.

[0065]FIG. 3 is a graph which depicts a MALDI-TOF timecourse massspectrograph of the deacetylation activity (−42 Da) of Hos3 enzyme onhistone H4(1-24).

[0066]FIG. 4 is a graph which depicts IC₅₀ values of histone H4 (1-24)acetylated peptide, Trichostatin A, and sodium butyrate.

[0067]FIG. 5A is a graph which depicts Trichostatin A IC₅₀ valuesplotted as a function of substrate 7AC concentration.

[0068]FIG. 5B is a graph which depicts the Henderson plot of K_(i) forTrichostatin A.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0069] “Histone deacetylase” and “HDAC” refer to any one of a family ofenzymes that remove acetyl groups from the ε-amino groups of lysineresidues at the -terminus of a histone. Unless otherwise indicated, theterm “histone” is meant to refer to any histone protein, including H1,H2A, H2B, H3, H4, and H5, from any species. Preferred histonedeacetylases include Class I and Class II HDAC enzymes.

[0070] “Histone deacetylase inhibitor” or “inhibitor of histonedeacetylase” refers to a compound which is capable of interacting with ahistone deacetylase and inhibiting its enzymatic activity. Inhibitinghistone deacetylase enzymatic activity means reducing the ability of ahistone deacetylase to remove an acetyl group from a histone.

[0071] “Test sample” refers to a sample which contains a test compoundor natural product extract to be tested for histone deacetylase enzymeactivity.

[0072] “Alkenyl” means a branched or unbranched unsaturated hydrocarbonchain comprising a designated number of carbon atoms. For example, C₂-C₆straight or branched alkenyl hydrocarbon chain contains 2 to 6 carbonatoms having at least one double bond, and includes but is not limitedto substituents such as ethenyl, propenyl, iso-propenyl, butenyl,iso-butenyl, tert-butenyl, -pentenyl, -hexenyl, and the like. It is alsocontemplated as within the scope of the present invention that “alkenyl”may also refer to an unsaturated hydrocarbon chain wherein any of thecarbon atoms of said alkenyl are optionally replaced with O, NH, S, orSO₂. For example, carbon 2 of 4-pentene can be replaced with O to form(2-propene)oxymethyl.

[0073] “Alkoxy” refers to the group —OR wherein R is alkyl as hereindefined. Preferably, R is a branched or unbranched saturated hydrocarbonchain containing 1 to 6 carbon atoms.

[0074] “Alkyl” means a branched or unbranched saturated hydrocarbonchain comprising a designated number of carbon atoms. For example, C₁-C₆straight or branched alkyl hydrocarbon chain contains 1 to 6 carbonatoms, and includes but is not limited to substituents such as methyl,ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, N-pentyl,N-hexyl, and the like. It is also contemplated as within the scope ofthe present invention that “alkyl” may also refer to a hydrocarbon chainwherein any of the carbon atoms of said alkyl are optionally replacedwith O, NH, S, or SO₂. For example, carbon 2 of -pentyl can be replacedwith O to form propyloxymethyl.

[0075] Throughout this application, “R” or “R_(n)”, where n is a number,is used to designate various substituents. These R groups areindependently selected. Thus, for example, the fact that R₁ may be abranched alkyl in one context does not require that R₁ be the samebranched alkyl, and does not prohibit that R₁ be, for example, astraight chain alkenyl in another context in the same molecule. It isintended that all “R_(n)” are selected independently of all other“R_(n)”, whether or not the term “independently selected” is used.

[0076] “Aryl” or “aromatic” refers to an aromatic carbocyclic orheterocyclic group having a single ring, for example a phenyl ring;multiple rings, for example biphenyl; or multiple condensed rings inwhich at least one ring is aromatic, for example naphthyl,1,2,3,4-tetrahydronaphthyl, anthryl, or phenanthryl. The ring(s) of anaryl moiety can be unsubstituted or substituted with one or moresubstituents including, but not limited to, halo, hydroxyl, nitro,trifluoromethyl, C₁-C₆ straight or branched chain alkyl or alkenyl,C₁-C₄ alkoxy, C₁-C₄ alkenyloxy, phenoxy, benzyloxy, or amino; aheterocyclic ring may contain 1-6 heteroatom(s) selected from the groupconsisting of O, N, and S. The substituents attached to a phenyl ringportion of an aryl moiety in the compounds of the invention may beconfigured in the ortho-, meta-, or para- orientation(s), with the para-orientation being preferred.

[0077] Examples of typical aryl moieties included in the scope of thepresent invention may include, but are not limited to, the following:

[0078] It should be kept in mind that, throughout this application, “Ar”is used to designate various substituents. As indicated throughout,these Ar groups are independently selected. Thus, for example, the factthat Ar may be phenyl in one context does not require that Ar be phenyl,nor prohibit that Ar be, for example, pyridyl in another context in thesame molecule. It is intended that all “Ar” are selected independentlyof all other “Ar”, whether or not the term “independently selected” isused.

[0079] “Carbocycle” or “carbocyclic” refers to an organic cyclic moietyin which the cyclic skeleton is comprised of only carbon atoms, whereasthe term “heterocycle” or “heterocyclic” refers to an organic cyclicmoiety in which the cyclic skeleton contains one or more heteroatomsselected from nitrogen, oxygen, or sulfur, and which may or may notinclude carbon atoms. The term “carbocycle” refers to a carbocyclicmoiety containing the indicated number of carbon atoms. The term “C₃-C₈cycloalkyl”, therefore, refers to an organic cyclic substituent in whichthree to eight carbon atoms form a three, four, five, six, seven, oreight-membered ring, including, for example, a cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl ring.

[0080] “Carbocyclic” or “heterocyclic” each includes within its scope asingle ring system, multiple fused rings (for example, bicyclic,tricyclic, or other similar bridged ring systems or substituents, e.g.adamantyl) or multiple condensed ring systems. One skilled in the art,therefore, will appreciate that in the context of the present invention,a cyclic structure may comprise bi-, or tri-, or multiple condensedrings, bridged ring systems, or combinations thereof.

[0081] “Halo” refers to fluoro, chloro, bromo or iodo, unless otherwiseindicated.

[0082] “Heterocycle” or “heterocyclic”, refers to a saturated,unsaturated, or aromatic carbocyclic group having a single ring,multiple fused rings (for example, bicyclic, tricyclic, or other similarbridged ring systems or substituents), or multiple condensed rings, andhaving at least one heteroatom such as nitrogen, oxygen, or sulfurwithin at least one of the rings. This term also includes “Heteroaryl,”which refers to a heterocycle in which at least one ring is aromatic.Any heterocyclic or heteroaryl group can be unsubstituted or optionallysubstituted with one or more groups, as defined above. Further, bi- ortricyclic heteroaryl moieties may comprise at least one ring which iseither completely or partially saturated.

[0083] As one skilled in the art will appreciate, such heterocyclicmoieties may exist in several isomeric forms, all of which areencompassed by the present invention. For example, a 1,3,5-triazinemoiety is isomeric to a 1,2,4-triazine group. Such positional isomersare to be considered within the scope of the present invention.Likewise, the heterocyclic or heteroaryl groups can be bonded to othermoieties in the compounds of the present invention. The point(s) ofattachment to these other moieties is not to be construed as limiting onthe scope of the invention. Thus, by way of example, a pyridyl moietymay be bound to other groups through the 2-, 3-, or 4-position of thepyridyl group. All such configurations are to be construed as within thescope of the present invention.

[0084] Examples of heterocyclic or heteroaryl moieties included in thescope of the present invention may include, but are not limited to, thefollowing:

Methods of the Present Invention

[0085] DNA associated with histone proteins is organized intonucleosomes, which are then arranged into higher-order structures knownas chromatin. The N-terminal tails of histone proteins are the sites ofmany different types of modifications, including acetylation,phosphorylation, ubiquitination, and methylation. Acetylation of theε-amino group of specific lysine residues achieved by histoneacetyltransferase enzymes typically results in transcriptionalactivation and gene expression. On the other hand, hypoacetylationmediated by histone deacetylase enzymes is associated with repression ofgene expression and the maintenance of transcriptionally silentchromatin.

[0086] In addition to deacetylating histone tails, transcription factorssuch as p53, GATA-1, TFIIE, and TFIIF are HDAC substrates. HDACsparticipate in cell cycle control and growth regulation, andconsequently inhibitors of HDACs have demonstrated the ability to arresttumor cell growth, induce differentiation, and cause apoptosis. Agrowing body of experimental evidence indicates that the activity ofHDAC affects cell cycle arrest, terminal differentiation of differentcell types, and the pathogenesis of malignant disease. HDACs playintegral roles in cancer gene regulation and cancer proliferation andHDAC inhibitors represent promising new chemotherapies. For example, therole of HDAC has been highlighted by the finding that mutant retinoidreceptors recruit HDAC in acute promyelocytic leukemia. Thus, a thoroughcharacterization of HDAC activity is important not only to ourunderstanding of gene expression, but to our understanding of cancerpathology.

[0087] Interestingly, HDAC inhibitors such as trichostatin A andtrapoxin have been shown to induce cell differentiation, cell cyclearrest, and reversal of transformed cell morphology. Not surprisingly,therefore, a number of HDAC inhibitors show promise as antitumor agents.The discovery of antimalarial effects of certain HDAC inhibitors furthersupports the idea that HDACs will be key targets for chemotherapeuticintervention in a variety of human diseases.

[0088] Histone deacetylases regulate chromatin remodeling andtranscriptional silencing by removing acetyl groups from conservedlysine residues located on the N-terminal tails of histones. It isbelieved that histone deacetylases interact with, and are recruited bytumor suppressors and oncogenes. As a result, natural product inhibitorsof the histone deacetylase function, such as sodium butyrate andhydroxamic acids, are expected to be effective therapeutic agents forthe treatment of human cancers. Characterization of HDAC activity haspreviously been problematic due to a lack of pure, active enzyme and asuitable activity assay.

[0089] Fluorescence-based biological assays have been widely used forhigh-throughput screening for inhibitors of pharmaceutically interestingtarget enzymes. Suitable assay formats, however, have not yet beendeveloped for all important targets. For HDACs, the most frequently usedassay type is still isotopic and nonhomogeneous in nature. Homogeneousfluorogenic assays are highly desirable because a reaction product isreleased as a fluorescent moiety, distinguishable from the reactionsubstrate, during the enzymatic reaction.

[0090] The inventive subject matter provides in vitro monitoring of HDACactivity via fluorogenic assay. The inventive HDAC substrates contain anacetylated coumarin moiety which is optionally substituted. We havesynthesized an exemplary compound, 7-Acetoxycoumarin (hereinafter“7AC”), and employed it as a small molecule substrate for continuousmonitoring of HDAC activity in the first continuous fluorescent assayfor histone deacetylase activity. The inventive assay is nonradioactive,highly sensitive, and does not demand the consumption of expensivematerial such as histones. Experiments with Trichostatin A and sodiumbutyrate, known inhibitors of HDAC, indicate that the new assay is wellsuited for high-throughput screening efforts for identifying novel HDACinhibitors from collections of candidate compounds and compositions.

[0091] It is expected that the inventive subject matter will greatlystreamline the process of selecting HDAC inhibitors and make morefeasible the identification of inhibitors targeted to specific HDACs.Thus, inhibition of a specific HDAC within an organism is expected tomake possible the targeted inhibition of HDAC enzyme(s) responsible fora specific disorder, such as an uncontrolled proliferation disorder.Similarly, inhibition of an HDAC of a specific organism is expected tomake possible the targeted inhibition of that organism, such as is foundin a protozoal or fungal infection.

Method for Screening for Histone Deacetylase Activity

[0092] The present invention relates to a method for screening forhistone deacetylase enzyme activity in a test sample, comprising thesteps of:

[0093] i) contacting said test sample with a liquid screening solutioncomprising a compound of Formula I:

[0094]  wherein:

[0095] R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of:

[0096] hydrogen,

[0097] hydroxyl,

[0098] carbonyl,

[0099] acetyl,

[0100] Ar,

[0101] straight or branched chain C₁-C₉ alkyl,

[0102] straight or branched chain C₁-C₉ alkyl substituted with one ormore halo, trifluoromethyl, nitro, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy,C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0103] straight or branched chain C₂-C₉ alkenyl or alkynyl, and

[0104] straight or branched chain C₂-C₉ alkenyl or alkynyl substitutedwith one or more halo, trifluoromethyl, nitro, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy,C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0105] provided that at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈must be —O—C(O)—R₉, wherein R₉ is straight or branched chain C₁-C₃alkyl; and

[0106] Ar is a mono-, bi- or tricyclic, carbo- or heterocyclic ring,wherein the ring is either unsubstituted or substituted in one or moreposition(s) with halo, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straightor branched chain alkyl or alkenyl, C₁-C₄ alkoxy, C₁-C₄ alkenyloxy,phenoxy, benzyloxy, or amino; wherein the individual ring sizes are 5-6members; and wherein the heterocyclic ring contains 1-6 heteroatom(s)selected from the group consisting of O, N, and S; and

[0107] ii) assaying for fluorescence produced by a product of histonedeacetylase enzyme activity in said liquid screening solution.

[0108] In another aspect of the inventive subject matter, said Ar isselected from the group consisting of naphthyl, indolyl, thioindolyl,furyl, thiazolyl, thienyl, pyridyl, quinolinyl, isoquinolinyl,fluorenyl, phenyl, and benzyl.

[0109] In a preferred embodiment, said Ar is phenyl.

[0110] In another aspect of the inventive subject matter, R₆ is—O—C(O)—R₉.

[0111] In a preferred embodiment, said R₆ is acetyl. 65

[0112] In a more preferred embodiment, said R₁, R₂, R₃, R₄, R₅, P₇, andR₈ are independently selected from the group consisting of:

[0113] hydrogen,

[0114] hydroxyl,

[0115] carbonyl,

[0116] acetyl,

[0117] Ar,

[0118] straight or branched chain C₁-C₃ alkyl, and

[0119] straight or branched chain C₁-C₃ alkyl substituted with halo,trifluoromethyl, nitro, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄alkenyloxy, phenoxy, benzyloxy, amino, or Ar.

[0120] In another more preferred embodiment, said R₁, R₂, R₃, R₄, R₅,R₇, and R₈ are independently selected from the group consisting of:

[0121] hydrogen,

[0122] hydroxyl,

[0123] carbonyl,

[0124] acetyl,

[0125] Ar,

[0126] straight or branched chain C₁-C₃ alkyl, and

[0127] straight or branched chain C₁-C₃ alkyl substituted with halo,trifluoromethyl, nitro, hydroxy, phenoxy, benzyloxy, amino, or Ar.

[0128] In a further more preferred embodiment, said R₁, R₂, R₃, R₄, R₅,R₇, and R₈ are independently selected from the group consisting of:

[0129] hydrogen,

[0130] methyl,

[0131] hydroxyl,

[0132] carbonyl,

[0133] acetyl, and

[0134] phenyl.

[0135] In another aspect of the inventive subject matter, R₁, R₂, R₃,R₄, R₅, R₆, R₇, and R₈ are independently selected from the groupconsisting of:

[0136] hydrogen,

[0137] hydroxyl,

[0138] carbonyl,

[0139] acetyl,

[0140] Ar,

[0141] straight or branched chain C₁-C₃ alkyl, and

[0142] straight or branched chain C₁-C₃ alkyl substituted with halo,trifluoromethyl, nitro, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄alkenyloxy, phenoxy, benzyloxy, amino, or Ar.

[0143] In a particularly preferred embodiment, said compound is ofFormula II:

[0144] wherein X is selected from the group consisting of hydrogen,methyl, hydroxyl, carbonyl, acetyl, and phenyl.

[0145] In a most preferred embodiment, X is hydrogen or phenyl, and saidassay for fluorescence is conducted at about 447 nm.

Method for Screening for Histone Deacetylase Inhibitors

[0146] The present invention further relates to a method for screeningfor inhibitors of histone deacetylase enzyme activity, comprising thesteps of:

[0147] i) contacting an inhibitor candidate compound or composition witha liquid screening solution comprising a histone deacetylase enzyme anda compound of Formula I:

[0148]  wherein:

[0149] R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of:

[0150] hydrogen,

[0151] hydroxyl,

[0152] carbonyl,

[0153] acetyl,

[0154] Ar,

[0155] straight or branched chain C₁-C₉ alkyl,

[0156] straight or branched chain C₁-C₉ alkyl substituted with one ormore halo, trifluoromethyl, nitro, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy,C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0157] straight or branched chain C₂-C₉ alkenyl or alkynyl, and

[0158] straight or branched chain C₂-C₉ alkenyl or alkynyl substitutedwith one or more halo, trifluoromethyl, nitro, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy,C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar,

[0159] provided that at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈must be —O—C(O)—R₉, wherein R₉ is straight or branched chain C₁-C₃alkyl; and

[0160] Ar is a mono-, bi- or tricyclic, carbo- or heterocyclic ring,wherein the ring is either unsubstituted or substituted in one or moreposition(s) with halo, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straightor branched chain alkyl or alkenyl, C₁-C₄ alkoxy, C₁-C₄ alkenyloxy,phenoxy, benzyloxy, or amino; wherein the individual ring sizes are 5-6members; and wherein the heterocyclic ring contains 1-6 heteroatom(s)selected from the group consisting of O, N, and S; and

[0161] ii) assaying for fluorescence produced by a product of histonedeacetylase enzyme activity in said reaction solution.

[0162] In another aspect of the inventive subject matter, said Ar isselected from the group consisting of naphthyl, indolyl, thioindolyl,furyl, thiazolyl, thienyl, pyridyl, quinolinyl, isoquinolinyl,fluorenyl, phenyl, and benzyl.

[0163] In a preferred embodiment, said Ar is phenyl.

[0164] In another aspect of the inventive subject matter, R₆ is—O—C(O)—R₉.

[0165] In a preferred embodiment, said R₆ is acetyl.

[0166] In a more preferred embodiment, said R₁, R₂, R₃, R₄, R₅ R₇, andR₈ are independently selected from the group consisting of:

[0167] hydrogen,

[0168] hydroxyl,

[0169] carbonyl,

[0170] acetyl,

[0171] Ar,

[0172] straight or branched chain C₁-C₃ alkyl, and

[0173] straight or branched chain C₁-C₃ alkyl substituted with halo,trifluoromethyl, nitro, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄alkenyloxy, phenoxy, benzyloxy, amino, or Ar.

[0174] In another more preferred embodiment, said R₁, R₂, R₃, R₄, R₅,R₇, and R₈ are independently selected from the group consisting of:

[0175] hydrogen,

[0176] hydroxyl,

[0177] carbonyl,

[0178] acetyl,

[0179] Ar,

[0180] straight or branched chain C₁-C₃ alkyl, and

[0181] straight or branched chain C₁-C₃ alkyl substituted with halo,trifluoromethyl, nitro, hydroxy, phenoxy, benzyloxy, amino, or Ar.

[0182] In a further more preferred embodiment, said R₁, R₂, R₃, R₄, R₅,R₇, and R₈ are independently selected from the group consisting of:

[0183] hydrogen,

[0184] methyl,

[0185] hydroxyl,

[0186] carbonyl,

[0187] acetyl, and

[0188] phenyl.

[0189] In another aspect of the inventive subject matter, R₁, R₂, R₃,R₄, R₅, R₆, R₇, and R₈ are independently selected from the groupconsisting of:

[0190] hydrogen,

[0191] hydroxyl,

[0192] carbonyl,

[0193] acetyl,

[0194] Ar,

[0195] straight or branched chain C₁-C₃ alkyl, and

[0196] straight or branched chain C₁-C₃ alkyl substituted with halo,trifluoromethyl, nitro, C₁-C₆ straight or branched chain alkyl, C₂-C₆straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄alkenyloxy, phenoxy, benzyloxy, amino, or Ar.

[0197] In a particularly preferred embodiment, said compound is ofFormula II:

[0198] wherein X is selected from the group consisting of hydrogen,methyl, hydroxyl, carbonyl, acetyl, and phenyl.

[0199] In a most preferred embodiment, X is hydrogen or phenyl, and saidassay for fluorescence is conducted at about 447 nm.

[0200] The novel HDAC assay presented herein combines the specificity ofthe deacetylation reaction with the advantages of a homogeneousfluorogenic assay in a single-step process. The inventive HDACsubstrates produce a de-acetylated fluorophore which is detectable bystandard fluorescence measurements. The assay described here is wellsuited in the context of high-throughput screening for HDAC inhibitors,even in small reaction volumes.

[0201] It will be understood by one of ordinary skill in the art thatenzymes having HDAC activity generally function in coordinationcomplexes additionally comprising one or more divalent metal ion(s),particularly zinc. Thus, in the case of a recombinant enzyme forexample, it may be necessary for a sample to be tested for HDACactivity, or a screening solution for detecting inhibition of HDACactivity, to additionally comprise divalent metal ions. Exemplarydivalent metal ions, which may be required to form functional HDACenzyme-metal coordination complexes include, but are not limited to,zinc, copper, manganese, iron, nickel, calcium, and magnesium. Zinc andmanganese are the more preferred among such exemplary divalent metalions. We have found that manganese is particularly effective with theHos3 enzyme.

[0202] Spectrophotomeric HDAC Activity Assays. Three differentchromophores were utilized in developing the inventive subject matter:

[0203] As depicted in FIG. 1, the inventive fluorescence-based HDACassay monitors the hydrolysis of 7AC to 7-hydroxycoumarin, or of ACC to7-hydroxycoumarin-3-carboxylic acid, at λ_(ex)=332 nm and λ_(em)=447 nm.7AC has an advantage over ACC due to a lower background rate ofhydrolysis in the assay buffer at pH 8.0. Utilization rations for eachenzyme with the 7AC substrate were calculated. For Hos3, the utilizationratio was 1.9122×10⁻⁴ s⁻¹, and 1274.8 M⁻¹ s⁻¹ when normalized by enzymeconcentration. For HDLP, the utilization ratio was 2.0375×10⁻⁴ s⁻¹ and612.8 M⁻¹ s⁻¹ when normalized by enzyme concentration. For HDAC2, theutilization ratio was 1.2526×10⁻⁵ s⁻¹, and 35.8 M⁻¹ s⁻¹ when normalizedby enzyme concentration, as shown in Table 1. TABLE 1 Utilization RatiosDetermined Using 7AC Substrate Enzyme V/K (s⁻¹) V/[E_(t)]/K (M⁻¹ s⁻¹)Hos3 1.9122 × 10⁻⁴ 1274.8 HDLP 2.0375 × 10⁻⁴ 612.8 HDAC2 1.2526 × 10⁻⁵35.8

[0204] The substrates 7AC and ACC can also be effectively utilized in aUV assay. The substrate absorbs from 250-340 nm, while the product has amaximal absorbance at 375 nm. So substrate hydrolysis/product formationcan be monitored by the increase in absorbance at 375 nm. For Hos3, theK_(m) ^(app) with 7AC using the UV assay was 649 mM. The K_(m) ^(app)may be lower than the actual K_(m) due to the limited solubility of thesubstrate in the assay buffer at concentrations greater than 1 mM.Para-nitrophenylacetate can also be used to assay HDAC activity. Uponacetate hydrolysis, the para-nitrophenylacetate liberates the coloredp-nitrophenol chromophore; this reaction can be monitored on the UV at410 nm. Using MBP-Hos3, the K_(m) ^(app) was determined to be 8.3 mM.This substrate is also subject to solubility limitations that preventcomplete saturation of the enzyme.

[0205] Protein Expression and Purification. FIG. 2A shows MBP-Hos3contains an internal TEV protease site for select proteolytic removal ofthe MBP fusion protein. Similarly, FIG. 2B shows an expansion of thejunction between Hos3 and MBP showing the TEV protease cleavage site. Asdemonstrated in FIGS. 2A and 2B, the Hos3 gene was successfully clonedinto the pMBP parallel2 vector to produce pMBPTEVHOS3. The MBP fusionprotein was then overexpressed in E.coli BL21 (DE3) cells. As shown inFIG. 2C, purification resulted in a final yield of approximately 20 mgHos3 protein per liter cells. In FIG. 2C, lane 1: Affinity-purifiedMBP-Hos3; lane 2: Heparin-agarose purified Hos3; remaining lane:molecular weight standards.

[0206] Characterization of Hos3 Activity by Mass Spectrometry. Hos3demonstrated the ability to deacetylate a hyperacetylated histoneH4(1-23) substrate in vitro. 1 uM MBP-Hos3 enzyme was incubated with 100uM of histone H4(1-24) acetylated peptide substrate for 240 minutes inbuffer A. Aliquots removed during the time course were subjected toMALDI-TOF mass spectrometry for mass determination. Aliquots wereremoved at specific time points and quenched with 0.01%TFA and desaltedby HPLC. As shown in FIG. 3, all acetylated positions, Lys 5, 8, 12, 16,and 20 were deacetylated by MBP-Hos3.

[0207] Characterization of the Mechanism of Inhibition. Inhibitorstudies were conducted using the 7AC substrate and the fluorescenthistone deacetylase activity assay. As shown in Table 2 and FIG. 4, IC₅₀values of Hos3 (150 nM) with 7AC (75 mM) substrate and alternatesubstrate histone H4 (1-24) acetylated peptide, and inhibitorsTrichostatin A and sodium butyrate, were determined using ourfluorescent assay. The mixed-competitive inhibitor sodium butyratedisplayed an IC₅₀ of 13.4 (+3.8) mM and a K_(i) of 12 mM. TABLE 2 Modeof Inhibition IC₅₀ value K_(i) H4(1-24) Alternate Non-Competitive  51.2± 7 μM 50 μM Substrate NaB Inhibitor Mixed-Competitive  13.4 ± 3.8 mM 12mM TSA Inhibitor Tight-binding Comp 127.0 ± 11.0 nM 81 nM

[0208] Despite showing apparent non-competitive inhibitory patterns,Trichostatin A was proven to be a tight-binding competitive inhibitorwhen IC₅₀ values were plotted as a function of substrate concentration,as shown in FIG. 5A. The K_(i)=81 nM was obtained from the slope of theline of the Henderson plot depicted in FIG. 5B. AS shown in Table 2 andFIGS. 5A and 5B, Trichostatin A is a tight-binding competitive inhibitorwith an IC₅₀ value of 127.0 (±11.0) nM and a K_(i) of 81 nM.

[0209] Characterization of an Alternate Substrate. Eitherspectrophotomeric HDAC assay can be used to screen alternate substratesby competition. As shown in Table 2, the IC₅₀ and K_(i) values foralternate substrate histone H4(1-24)Ac were determined to be 51.2 (±7)μM and 50 μM, respectively.

[0210] Using the inventive substrates, kinetic parameters andutilization ratios for each of three representative Class I/IIzinc-dependent HDAC enzymes, Saccharomyces cerevisiae Hos3, human HDAC2,and Aquifex aeolicus HDLP, were determined. Using 7AC, we determinedthat Trichostatin A, a hydroxamate inhibitor that previously was thoughtto exhibit non-competitive inhibitory patterns with acetylated histones,is actually a tight-binding, competitive inhibitor with a K_(i) value of81 nM and an IC₅₀ value of 127±11 nM. Sodium butyrate displaysmixed-type inhibition with a K_(i) value of 12 mM and an IC₅₀ of13.4±3.8 mM. Hos3 exhibited non-competitive inhibition with alternatesubstrate acetylated histone peptide H4 (1-24) with a K_(i) value of 50mM and an IC₅₀ value of 51.2±7 μM. Based on these studies, we expectbroad utility of using acetoxycoumarins as fluorogenic substrates forassaying for HDAC activity. The discovery of a small moleculefluorescent substrate for HDACs now makes more feasible the design andhigh-throughput evaluation of inhibitors for identification of cancerchemotherapeutic agents.

Synthesis of Compounds of the Invention

[0211] The inventive compounds may be readily prepared by standardtechniques of organic chemistry. In the preparation of the compounds ofthe invention, one skilled in the art will understand that one may needto protect or block various reactive functionalities on the startingcompounds or intermediates while a desired reaction is carried out onother portions of the molecule. After the desired reactions arecomplete, or at any desired time, normally such protecting groups willbe removed by, for example, hydrolytic or hydrogenolytic means. Suchprotection and deprotection steps are conventional in organic chemistry.One skilled in the art is referred to “Protective Groups in OrganicChemistry,” McOmie, ed., Plenum Press, New York, N.Y.; and “ProtectiveGroups in Organic Synthesis,” Greene, ed., John Wiley & Sons, New York,N.Y. (1981) for the teaching of protective groups which may be useful inthe preparation of compounds of the present invention.

[0212] The product and intermediates may be isolated or purified usingone or more standard purification techniques, including, for example,one or more of simple solvent evaporation, recrystallization,distillation, sublimation, filtration, chromatography, includingthin-layer chromatography, HPLC (eq. reverse phase HPLC), columnchromatography, flash chromatography, radial chromatography,trituration, and the like.

EXAMPLES

[0213] The following examples are illustrative of the present inventionand are not intended to be limitations thereon. Unless otherwiseindicated, all percentages are based upon 100% by weight of the finalcomposition.

[0214] DNA contained in the plasmid pHOS3.S2 plasmid DNA was generouslyprovided by Prof. Michael Grunstein from University of California at LosAngeles. Human HDAC2 clone as the GST-HDAC2 was generously provided byProf. Edward Seto from the University of Florida. GST-HDLP wasgenerously provided by Prof. Nikola Pavletich at Memorial SloanKettering Cancer Center. PMBP-parallel 2 vector was obtained from Prof.Patrick Loll at Drexel University. Dideoxy DNA sequencing was performedby the University of Pennsylvania Molecular Biology DNA Sequencing CoreFacility.

[0215] Oligonucleotide primers, electrocompetent E.coli BL21 (DE3)cells, tobacco etch virus (TEV) protease, ampicillin, heparin agarose,glutathione, Luria broth, isopropyl-β-D-thiogalactopyranoside, amyloseresin, glutathione sepharose, Trichostatin A,7-acetoxycoumarin-3-carboxylic acid, and para-nitrophenylacetate wereobtained from publicly available, commercial sources and are known tothose of ordinary skill in the art. Other reagents and solvents werepurchased from publicly available commercial sources and used withoutfurther purification.

[0216] Abbreviations used in the Examples are the following: “HDAC”refers to histone deacetylase; “HAT” refers to histone acetyltransferase; “Ac” refers to acetyl; “ACC” refers to7-acetoxycoumarin-3-carboxylic acid; “7AC” refers to 7-acetoxycoumarin;“pNPA” refers to 4-nitrophenylacetate; “TSA” refers to Trichostatin A;“IPTG” refers to isopropyl-b-D-thiogalactoside; “MBP” refers tomaltose-binding protein; “GST” refers to glutathione-s-transferase; “LB”refers to Luria-Burtani media; “PCR” refers to polymerase chainreaction; “SDS” refers to sodium dodecyl sulfate; “PAGE” refers topolyacrylamide gel electrophoresis; “DMSO” refers to dimethylsulfoxide;“K_(m)” refers to Michaelis constant; “k_(cat)” refers to catalyticturnover number; “K_(I)” refers to the dissociation constant of theinhibitor; “IC₅₀” refers to 50% inhibitory concentration; “ESI-FTMS”refers to high resolution Fourier transform electrospray massspectrometry; “MALDI-TOF” refers to matrix assisted laser desorptionionization time-of-flight mass spectrometry; “TEV” refers to tobaccoetch virus.

EXAMPLE 1 Synthesis of 7-Acetoxycoumarin (7AC)

[0217] The following example illustrates the preparation of a preferredHDAC substrate provided according to the present inventive subjectmatter.

[0218] To an ice cold solution of 20% acetic anhydride in pyridine (60ml) was added 25 mmol of 7-hydroxycoumarin (Aldrich) with stirring sothe temperature did not rise above 4° C. The reaction immediatelydarkened, stirring was continued at 0° C. for 30 min, the reaction wasallowed to warn to room temperature, and stirred an additional 2.5 h.The reaction mixture was rotary evaporated to dryness and the residuewas taken up in 100 ml EtOAc and washed with 10% citric acid (5×100 ml),brine (5×100 ml) dried with MgSO4 and evaporated to dryness. Theresulting solid was recrystallized two times from EtOAc/hexanes toprovide the title compound as a white solid: m.p. 135-137° C.; ¹H NMRd(ppm) 2.3 (s, 3H, CH₃), 6.4 (d, 1H, J=9.5 Hz, H2′), 7.1 (dd, 2H, J=1.5Hz, J=8.5 Hz, H3′, H2) 7.2 (d, 2H, J=1.5 Hz, H-2) 7.7 (d, 2H, J=9 Hz,H-5), 8.0 ppm (d, 2H, J=9.5 Hz, H-6); ¹³C NMRδ=20.721, 109.969, 115.395,116.512, 118.504, 129.188, 143.647, 152.777, 153.967, 159.588, 168.670ppm.

Example 2 Cloning, Overexpression and Purification of Hos3

[0219] Using PCR oligonucleotide primers derived from the gene sequence,the Saccharomyces cerevisiae Hos3 gene from pHOS3.S2 plasmid DNA wassubcloned into the Bam HI/HindIII sites of plasmid MBP-2 of pMBPparallel 2 to produce pMBPTEVHOS3. This vector is a T7 promoter-basedexpression vector that produces Hos3 N-terminally fused to MBP, with anintervening TEV protease recognition site for fusion protein removalafter affinity purification. The integrity of the clone was establishedby DNA sequencing. The pMBPTEVHOS3 plasmid was subsequently transformedinto BL21(DE3) E.coli electrocompetent cells for overexpression.Bacterial cultures were grown in LB media containing 200 μg/mlampicillin to an optical density of 0.8 at 1=595 nM. cells were inducedwith 1 mM IPTG and grown for 4 hours at 37° C. with shaking (200 rpm)then harvested by centrifugation (3000×g, 4° C., for 10 minutes.)Pellets from 2 L cells were resuspended in Buffer A (20 mM Tris, pH 8.0and 200 mM NaCl), lysed in a French Pressure cell and centrifuged at150,000×g, 4° C., for 1 h. The supernatant was diluted to 50 ml andapplied to amylose resin (1 ml/min) previously equilibrated with BufferA. The MBP fusion protein was eluted from the amylose with Buffer B (20mM Tris, pH 8.0 and 200 mM NaCl, 10 mM maltose). Fractions containingthe desired protein were identified by SDS-PAGE (12% acrylamide),combined, and dialyzed overnight into Buffer A. The protein was adjustedto 2.0 mg/ml and the fusion tag was removed proteolytically by theaddition of TEV protease (1:8 dilution) for 5 hours at room temperature.The proteolysis reaction mixture was applied to heparin agarose,previously equilibrated with Buffer A. The resin was washed in Buffer A(3×10 ml) then cut Hos3 was eluted with Buffer C (20 mM Tris, pH 8.0 and500 mM NaCl). Purified Hos3 was confirmed by SDS-PAGE. Proteinconcentrations were determined using the von Hippel method. Proteinaliquots were then flash frozen in liquid nitrogen and stored at −80° C.until use.

Example 3 Overexpression and Purification of HDAC2 and HDLP

[0220]E. coli BL21 (DE3) cells harboring the GST-HDAC2 and GST-HDLPexpression plasmids were grown, expressed, and harvested in the samemanner as was performed for pMBPTEVHOS3. Cell pellets were resuspendedin Buffer A, lysed in a French Pressure cell, and centrifuged at 150,0004° C., for 1 hour. The supernatant was diluted with Buffer A andincubated with glutathione sepharose beads (10 ml) for 1 hour, loaded onto a gravity filtration column, and washed with 10 column volumes ofBuffer A. GST fusion proteins were eluted with Buffer D (20 mM Tris, pH8.0, 200 mM NaCl, 10 mM reduced glutathione). Fractions containingpurified GST proteins identified by SDS-PAGE were combined and dialyzedovernight into Buffer A. GST-HDAC2 and GST-HDLP were concentrated to 0.1and 2.3 mg/ml respectively, aliquoted, flash frozen in liquid nitrogen,and stored at −80° C.

Example 4 Hos3 Deacetylation of Hyperacetylated Histone H4(1-23)

[0221] The histone H4(1-23) peptide was synthesized by standard FMOCsolid-phase peptide synthesis methods and hyperacetylated at Lys 5, 8,12, 16, and 20 post-cleavage with acetic anhydride in DMF/triethylamine.To confirm that Hos3 exhibited histone deacetylase activity, weincubated the peptide (100 μM) for 240 minutes with MBP-Hos3 (1 μMenzyme); aliquots removed throughout the time course, desalted by HPLCand analyzed by matrix-assisted time of flight mass spectrometry (MALDITOF-MS). The starting hyperacetylated peptide mass was 2625.8 Da, andfor each loss of an acetyl group the mass decreased by approximately 42Da.

Example 5 Histone Deacetylase Fluorescent Activity Assay

[0222] HDAC activity assays were performed in HDAC assay buffer (20 mMTris, pH 8.0, 500 mM NaCl, 1 mM MnCl₂) to a final reaction volume of 1ml in quartz spectrophotometer cuvettes. Fluorescence spectrophotometrywas used to monitor ACC or 7AC hydrolysis (λ_(ex)=332 nm, λ_(em)=447nm). Internal fluorescence intensity calibrations were obtained usingquinine sulfate as known in the art. ACC or 7AC substrate (solubilizedin 100% DMSO) was added in the appropriate concentrations to the assaybuffer (1% final concentration of DMSO) and the basal rate of hydrolysiswas recorded. After 90 seconds, 150 nM Hos3 enzyme was added and thereaction was allowed to proceed for 600 seconds. Initial rates for Hos3activity were defined as the initial rate of the reaction with enzymeadded, minus the basal rate of substrate hydrolysis. Utilization ratiosfor Hos3 were calculated based on the adjusted initial rates. The datawas fit by the following expressions (equations 1-3):

v=V _(max) [S]/K _(m) +[S], where [S]<<K _(m)  (1)

giving v=V _(max) [S]/K _(m)  (2)

which rearranges to v/[S]=V _(max) /K _(m)  (3)

[0223] where V_(max) is the maximum reaction velocity, [S] is thesubstrate concentration, K_(m) is the Michaelis constant, andV_(max)/K_(m) is the utilization ratio (V/K). Since V is dependent onthe total concentration of enzyme [E_(t)], utilization ratios arenormalized by enzyme concentration and reported in terms of V/[Et]/K.For GST-HDLP and GST-HDAC2, assay conditions and procedures were thesame except 330 nM of GST-HDLP and 350 nM of GST-HDAC2 were added toeach respective reaction.

Example 6 UV-Based Histone Deacetylase Activity Assays

[0224] HDAC activity assays were performed in HDAC Assay Buffer to afinal reaction volume of 1 ml in quartz cuvettes. UV/Visspectrophotometer was used to monitor the rate of 7AC hydrolysis. The7AC substrate absorbs from 250-340 nm, while the product,7-hydroxycoumarin, has a maximal absorbance at 375 nm. Reactions wereinitiated with substrate and allowed to proceed for 300 seconds, whilemonitoring the increase in absorbance at 375 nm. Para-nitrophenylacetatewas also used to assay HDAC activity. Upon acetate hydrolysis, thepara-nitrophenylacetate liberated the colored p-nitrophenol chromophore;this reaction was monitored on the UV at 410 nm. Initial rates andutilization ratios were calculated as described above.

Example 7 HDAC Inhibitor Studies

[0225] Competition experiments were performed in HDAC assay buffer (20mM Tris, pH 8.0, 500 mM NaCl, 1 mM MnCl₂) to a final reaction volume of1 ml. Trichostatin A was solubilized in 100% DMSO, yielding a finalconcentration of 2% DMSO in this reaction. Sodium butyrate and H4 (1-24)Ac peptide were both dissolved in water. The final DMSO concentration inthese reactions was 1%. 7AC substrate and inhibitor were added in theappropriate concentrations to the assay buffer and the basal rate ofhydrolysis was recorded. Hos3 enzyme (150 nM) was added after 90 secondsand the reaction was allowed to proceed for 600 seconds. Initial ratedata was transformed into appropriate rates and dose response plots weregenerated. IC₅₀ values were calculated using the program Kaleidagraphwith equation 4:

v _(i) /v _(o)=1/1+([I]/IC ₅₀)  (4)

[0226] where v_(i) is the initial velocity in the presence of inhibitor,v_(o) is the initial velocity in the absence of inhibitor , and [I] isthe concentration of inhibitor. K_(i) values were determined by Dixonplots (ref). The modes of inhibition were determined bydouble-reciprocal or Lineweaver-Burk plots, where 1/v is plotted as afunction of 1/[S], using the programs of Cleland.

[0227] In the case of Trichostatin A, the double-reciprocal plot yieldedapparent non-competitive inhibition that was later determined to betight binding competitive inhibition by plotting IC₅₀ values as afunction of [S] substrate concentration, which resulted in a straightline with a positive slope. To get the K_(i) value, points between therange of v_(i)/v_(o)=0.2 to 0.9 from the dose-response curve were usedto generate a Henderson plot, where [I]/(1-v_(i)/v_(o)) is plotted as afunction of v_(i)/v_(o), with the slope of the line equal to K_(i)(1+[S]/K_(m)) and the y-intercept equal to the total enzymeconcentration [E_(t)].

[0228] The invention being thus described, it will be obvious that thesame may be modified or varied in many ways. Such modifications andvariations are not to be regarded as a departure from the spirit andscope of the invention and all such modifications and variations areintended to be included within the scope of the following claims.

We claim:
 1. A method for screening for histone deacetylase enzymeactivity in a test sample, comprising the steps of: i) contacting saidtest sample with a liquid screening solution comprising a compound ofFormula I:

 wherein: R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of: hydrogen, hydroxyl, carbonyl, acetyl, Ar,straight or branched chain C₁-C₉ alkyl, straight or branched chain C₁-C₉alkyl substituted with one or more halo, trifluoromethyl, nitro, C₁-C₆straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy,amino, or Ar, straight or branched chain C₂-C₉ alkenyl or alkynyl, andstraight or branched chain C₂-C₉ alkenyl or alkynyl substituted with oneor more halo, trifluoromethyl, nitro, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy,C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar, provided that atleast one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ must be —O—C(O)—R₉,wherein R₉ is straight or branched chain C₁-C₃ alkyl; and Ar is a mono-,bi- or tricyclic, carbo- or heterocyclic ring, wherein the ring iseither unsubstituted or substituted in one or more position(s) withhalo, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straight or branched chainalkyl or alkenyl, C₁-C₄ alkoxy, C₁-C₄ alkenyloxy, phenoxy, benzyloxy, oramino; wherein the individual ring sizes are 5-6 members; and whereinthe heterocyclic ring contains 1-6 heteroatom(s) selected from the groupconsisting of O, N, and S; and ii) assaying for fluorescence produced bya product of histone deacetylase enzyme activity in said liquidscreening solution.
 2. The method of claim 1, wherein said Ar isselected from the group consisting of naphthyl, indolyl, thioindolyl,furyl, thiazolyl, thienyl, pyridyl, quinolinyl, isoquinolinyl,fluorenyl, phenyl, and benzyl.
 3. The method of claim 2, wherein said Aris phenyl.
 4. The method of claim 1, wherein R₆ is —O—C(O)—R₉.
 5. Themethod of claim 4, wherein R₆ is acetyl.
 6. The method of claim 5,wherein R₁, R₂, R₃, R₄, R₅, R₇, and R₈ are independently selected fromthe group consisting of: hydrogen, hydroxyl, carbonyl, acetyl, Ar,straight or branched chain C₁-C₃ alkyl, and straight or branched chainC₁-C₃ alkyl substituted with halo, trifluoromethyl, nitro, C₁-C₆straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy,amino, or Ar.
 7. The method of claim 6, wherein R₁, R₂, R₃, R₄, R₅, R₇,and R₈ are independently selected from the group consisting of:hydrogen, hydroxyl, carbonyl, acetyl, Ar, straight or branched chainC₁-C₃ alkyl, and straight or branched chain C₁-C₃ alkyl substituted withhalo, trifluoromethyl, nitro, hydroxy, phenoxy, benzyloxy, amino, or Ar.8. The method of claim 7, wherein R₁, R₂, R₃, R₄, R₅, R₇, and R₈ areindependently selected from the group consisting of: hydrogen, methyl,hydroxyl, carbonyl, acetyl, and phenyl.
 9. The method of claim 1,wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of: hydrogen, hydroxyl, carbonyl, acetyl, Ar,straight or branched chain C₁-C₃ alkyl, and straight or branched chainC₁-C₃ alkyl substituted with halo, trifluoromethyl, nitro, C₁-C₆straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy,amino, or Ar.
 10. The method of claim 1, wherein said compound is ofFormula II:

wherein X is selected from the group consisting of hydrogen, methyl,hydroxyl, carbonyl, acetyl, and phenyl.
 11. The method of claim 10,wherein X is hydrogen or phenyl, and said assay for fluorescence isconducted at about 447 nm.
 12. A method for screening for inhibitors ofhistone deacetylase enzyme activity, comprising the steps of: i)contacting an inhibitor candidate compound or composition with a liquidscreening solution comprising a histone deacetylase enzyme and acompound of Formula I:

 wherein: R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of: hydrogen, hydroxyl, carbonyl, acetyl, Ar,straight or branched chain C₁-C₉ alkyl, straight or branched chain C₁-C₉alkyl substituted with one or more halo, trifluoromethyl, nitro, C₁-C₆straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy,amino, or Ar, straight or branched chain C₂-C₉ alkenyl or alkynyl, andstraight or branched chain C₂-C₉ alkenyl or alkynyl substituted with oneor more halo, trifluoromethyl, nitro, C₁-C₆ straight or branched chainalkyl, C₂-C₆ straight or branched chain alkenyl, hydroxy, C₁-C₄ alkoxy,C₂-C₄ alkenyloxy, phenoxy, benzyloxy, amino, or Ar, provided that atleast one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ must be —O—C(O)—R₉,wherein R₉ is straight or branched chain C₁-C₃ alkyl; and Ar is a mono-,bi- or tricyclic, carbo- or heterocyclic ring, wherein the ring iseither unsubstituted or substituted in one or more position(s) withhalo, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straight or branched chainalkyl or alkenyl, C₁-C₄ alkoxy, C₁-C₄ alkenyloxy, phenoxy, benzyloxy, oramino; wherein the individual ring sizes are 5-6 members; and whereinthe heterocyclic ring contains 1-6 heteroatom(s) selected from the groupconsisting of O, N, and S; and ii) assaying for fluorescence produced bya product of histone deacetylase enzyme activity in said reactionsolution.
 13. The method of claim 12, wherein said Ar is selected fromthe group consisting of naphthyl, indolyl, thioindolyl, furyl,thiazolyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, fluorenyl,phenyl, and benzyl.
 14. The method of claim 13, wherein said Ar isphenyl.
 15. The method of claim 12, wherein R₆ is —O—C(O)—R₉.
 16. Themethod of claim 15, wherein R₆ is acetyl.
 17. The method of claim 16,wherein R₁, R₂, R₃, R₄, R₅, R₇, and R₈ are independently selected fromthe group consisting of: hydrogen, hydroxyl, carbonyl, acetyl, Ar,straight or branched chain C₁-C₃ alkyl, and straight or branched chainC₁-C₃ alkyl substituted with halo, trifluoromethyl, nitro, C₁-C₆straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy,amino, or Ar.
 18. The method of claim 17, wherein R₁, R₂, R₃, R₄, R₅,R₇, and R₈ are independently selected from the group consisting of:hydrogen, hydroxyl, carbonyl, acetyl, Ar, straight or branched chainC₁-C₃ alkyl, and straight or branched chain C₁-C₃ alkyl substituted withhalo, trifluoromethyl, nitro, hydroxy, phenoxy, benzyloxy, amino, or Ar.19. The method of claim 18, wherein R₁, R₂, R₃, R₄, R₅, R₇, and R₈ areindependently selected from the group consisting of: hydrogen, methyl,hydroxyl, carbonyl, acetyl, and phenyl.
 20. The method of claim 12,wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selectedfrom the group consisting of: hydrogen, hydroxyl, carbonyl, acetyl, Ar,straight or branched chain C₁-C₃ alkyl, and straight or branched chainC₁-C₃ alkyl substituted with halo, trifluoromethyl, nitro, C₁-C₆straight or branched chain alkyl, C₂-C₆ straight or branched chainalkenyl, hydroxy, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy, phenoxy, benzyloxy,amino, or Ar.
 21. The method of claim 12, wherein said compound is ofFormula II:

wherein X is selected from the group consisting of hydrogen, methyl,hydroxyl, carbonyl, acetyl, and phenyl.
 22. The method of claim 21,wherein X is hydrogen or phenyl, and said assay for fluorescence isconducted at about 447 nm.